Cancer or pre-cancerous growth generally refers to malignant tumors, rather than benign tumors. Benign tumor cells are similar to normal, surrounding cells. Treatment becomes necessary only when the tumors grow large enough to interfere with other organs. Malignant tumors, by contrast, grow faster than benign tumors, and they penetrate and destroy local tissues. Some malignant tumors may spread throughout the body via blood or the lymphatic system. The unpredictable and uncontrolled growth makes malignant cancers dangerous, and fatal in many cases. These tumors are not morphologically typical of the original tissue and are not encapsulated. Malignant tumors commonly recur after surgical removal.
Many human diseases are a result of proliferative cellular pathologies. Cancer or pre-cancerous growth is frequently a consequence of proliferative cellular pathologies and generally refers to malignant tumors, rather than benign tumors. Benign tumor cells are similar to normal, surrounding cells. Treatment becomes necessary only when the tumors grow large enough to interfere with other organs. Malignant tumors, by contrast, grow faster than benign tumors, and they penetrate and destroy local tissues. Some malignant tumors may spread throughout the body via blood or the lymphatic system, and their unpredictable and uncontrolled growth makes malignant cancers dangerous, and fatal in many cases. Such tumors are not morphologically typical of the original tissue and are not encapsulated. Malignant tumors commonly recur after surgical removal. Accordingly, treatment of proliferative diseases ordinarily targets proliferative cellular activities such as occur in malignant cancers or malignant tumors with a goal to intervene in the proliferative processes.
The inhibition or prevention of malignant growth is most effective at the early stage of the cancer development. It is important, therefore, to identify and validate molecular targets that play a role in proliferative processes and their induction and, in malignant diseases in particular, early signs of tumor formation. A particular goal is to determine potent tumor growth or gene expression suppression elements or agents associated therewith. The development of such tumor growth and/or gene expression and therapeutic elements or agents involves an understanding of the genetic control mechanisms for cell division and differentiation, particularly in connection with tumorigenesis. Unfortunately, the number of established protein targets that are suitable for intervention in proliferative disease is limiting. Of the small number of established targets, such as growth factors like EGF and its receptor, few, if any, permit adequate intervention in proliferative diseases such as malignant cancer and psoriasis.
Moreover, it has proven difficult to identify better targets for intervention in cellular proliferative pathologies. Large numbers of genes and proteins exist within the human genome and many of these genes and proteins, as well as post-translationally modified forms of the proteins, correlate with cellular proliferative pathologies. Of these many genes, proteins, and post-translationally modified proteins, only a few specific factors can be targeted to effectively intervene in cellular proliferative pathologies. Therefore, identification of these specific factors is needed. In addition to a need to identify specific genes, proteins, and post-translationally modified proteins to target to intervene in proliferative cellular pathologies, another problem is a need to confirm that the targeted factor indeed provides effective intervention within the active pathology within active pathological tissues. Unfortunately, proliferation of cells in cell culture conditions shows many factors can be targeted but most ultimately do not prove effective as intervention targets in active pathological tissues. Consequently, accurate identification of targets for effective intervention in proliferative cellular pathologies requires study of active pathological tissues such as in animal models of human disease.
Accordingly, treatment ordinarily targets malignant cancers or malignant tumors. The intervention of malignant growth is most effective at the early stage of the cancer development. It is thus exceedingly important to identify and validate a target for early signs of tumor formation and to determine potent tumor growth or gene expression suppression elements or agents associated therewith. The development of such tumor growth and/or gene expression and therapeutic elements or agents involves an understanding of the genetic control mechanisms for cell division and differentiation, particularly in connection with tumorigenesis.
RNA interference (RNAi) is a post-transcriptional process where the double-stranded RNA (dsRNA) inhibits gene expression in a sequence specific fashion. The RNAi process occurs in at least two steps: in first step, the longer dsRNA is cleaved by an endogenous ribonuclease into shorter, less than 100-, 50-, 30-, 23-, or 21-nucleotide-long dsRNAs, termed “small interfering RNAs” or siRNAs. In second step, the smaller siRNAs mediate the degradation of the target mRNA molecule. This RNAi effect can be achieved by introducing either longer dsRNA or shorter siRNA to the target sequence within cells. It is also demonstrated that RNAi effect can be achieved by introducing plasmids that generate dsRNA complementary to target gene.
The RNAi have been sucessfully used in gene function determination in Drosophila (Kennerdell et al. (2000) Nature Biotech 18: 896-898; Worby et al. (2001) Sci STKE Aug. 14, 2001 (95):PL1; Schmid et al. (2002) Trends Neurosci 25(2):71-74; Hammond et al. (2000). Nature, 404: 293-298), C. elegans (Tabara et al. (1998) Science 282: 430-431; Kamath et al. (2000) Genome Biology 2: 2.1-2.10; Grishok et al. (2000) Science 287: 2494-2497), and Zebrafish (Kennerdell et al. (2000) Nature Biotech 18: 896-898). In those model organisms, it has been reported that both the chemically synthesized shorter siRNA or in vitro transcripted longer dsRNA can effectively inhibit target gene expression. There are increasing reports on successfully achieved RNAi effects in non-human mammalian and human cell cultures (Manche et al. (1992). Mol. Cell. Biol. 12:5238-5248; Minks et al. (1979). J. Biol. Chem. 254:10180-10183; Yang et al. (2001) Mol. Cell. Biol. 21(22):7807-7816; Paddison et al. (2002). Proc. Natl. Acad. Sci. USA 99(3):1443-1448; Elbashir et al. (2001) Genes Dev 15(2):188-200; Elbashir et al. (2001) Nature 411: 494-498; Caplen et al. (2001) Proc. Natl. Acad. Sci. USA 98: 9746-9747; Holen et al. (2002) Nucleic Acids Research 30(8):1757-1766; Elbashir et al. (2001) EMBO J 20: 6877-6888; Jarvis et al. (2001) TechNotes 8(5): 3-5; Brown et al. (2002) TechNotes 9(1): 3-5; Brummelkamp et al. (2002) Science 296:550-553; Lee et al. (2002) Nature Biotechnol. 20:500-505; Miyagishi et al. (2002) Nature Biotechnol. 20:497-500; Paddison et al. (2002) Genes & Dev. 16:948-958; Paul et al. (2002) Nature Biotechnol. 20:505-508; Sui et al. (2002) Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu et al. (2002) Proc. Natl. Acad. Sci. USA 99(9):6047-6052).
EGFR-RP (Validated Target ICT1024): Homo sapiens Epithelial growth factor receptor-related protein, EGFR-RP or EGFR-RS is published GenBank accession nos. are AK026010, NM—022450, BC014425, AK056708 and M99624.
All eukaryotic cells contain elaborate systems of internal membranes which set up various membrane-enclosed compartments within the cell. The plasma membrane serves as the interface between the machinery in the interior of the cell and the extracellular fluid (ECF) that bathes all cells. Cell membranes are built from lipids and proteins. The lipids in the plasma membrane are chiefly phospholipids like phosphatidyl ethanolamine and cholesterol. Phospholipids are amphiphilic with the hydrocarbon tail of the molecule being hydrophobic; its polar head hydrophilic. As the plasma membrane faces watery solutions on both sides, its phospholipids accommodate this by forming a phospholipid bilayer with the hydrophobic tails facing each other. Many of the proteins associated with the plasma membrane are tightly bound to it. Some are attached to lipids in the bilayer, and others are transmembrane proteins —the polypeptide chain actually traverses the lipid bilayer.
All membrane proteins have a specific upside-down or right-side-up orientation in the bilayer. Some proteins are anchored to the membrane by ionic interactions between residues with positively charged side chains and negatively charged lipid head groups since biological membranes tend to have a net negative charge. Other proteins are anchored by post-synthetic attachment of a hydrocarbon chain such as myristoyl, palmitoyl, farnesyl or gerenyl-gerenyl, or a lipid such as glycosylphosphatidylinositol (GPI) which confines them in regions close to their protein partners. Other proteins are anchored to the surface by ionic contacts. The term monotopic or peripheral membrane protein refers to proteins that have a fairly shallow penetration of the membrane surface. Many peripheral proteins can be released from the membrane by increasing the ionic strength of the solution. A second category of membrane proteins is integral or transmembrane bitopic or multitopic proteins. These proteins can only be released from the membrane by bilayer disruption with detergents.
Many transmembrane proteins that are structurally related are also functionally related. For example, the EGF (epidermal growth factor receptor) and the insulin receptor fall into a family of growth factor receptors which have very large disulfide-rich extracellular and a tyrosine kinase intracellular domains connected by a single-transmembrane helix. Most members of this family are monomers and binding of ligand induces dimerization and activation of the intracellular tyrosine kinase domain. The insulin receptor is a dimer in its non-ligand bound state and it is possible that in this case the binding of insulin changes the intersubunit orientation of the monomers, allowing for activation.
Another important family of transmembrane proteins is the seven transmembrane family of G proteins (guanine nucleotide binding proteins) coupled receptors. These receptors are the most abundant class of receptors in mammalian cells and mediate an extremely diverse range of signals into the cell, from light (rhodopsin) to neurotransmitters (muscarinic or adrenergic receptors) to sex-related signals (oxytocin). Although their ligand activators are diverse, these receptors all couple to G proteins to transduce their signal. Structurally, they are similar in having seven transmembrane loops in a defined topology. In contrast to the growth factor receptor family, these proteins have relatively small extramembrane loops.
Integral membrane proteins that transport species such as nutrients and ions must be able to shield their ligands from the surrounding hydrocarbon interior. Thus, these proteins are much larger than the signal transduction proteins mentioned above, and often contain several subunits. An example of this class is the 12 membrane spanning family belonging to transporters, such as GLUT1 and antibiotics. A newly identified family of integral membrane proteins, Rhomboid family, is exemplified by the rhomboid (RHO) protein from Drosophila melanogaster, a developmental regulator involved in epidermal growth factor (EGF)-dependent signaling pathways (1, 2, 3). Not only were homologs of rhomboid detected in prokaryotes and eukaryotes, but the pattern of sequence conservation in this family appeared uncharacteristic of nonenzymatic membrane proteins, such as transporters (4, 5). Specifically, several polar amino-acid residues are conserved in nearly all members of the rhomboid family, suggesting the possibility of an enzymatic activity. As three of these conserved residues were histidines, it appears that rhomboid-family proteins may function as metal-dependent membrane proteases (5, 6). Recently, however, it has been shown that RHO cleaves a transmembrane helix (TMH) in the membrane-bound precursor of the TGFα-like growth factor Spitz, enabling the released Spitz to activate the EGF receptor, and that a conserved serine and a conserved histidine in RHO are essential for this cleavage (7, 8). Thus, it appears that rhomboid-family proteins are a distinct group of intramembrane serine proteases. Altogether, the genome of Drosophila encodes seven RHO paralogs (now designated RHO 1-7, with the original rhomboid becoming RHO-1), at least three of which are involved in distinct EGF-dependent pathways, apparently through proteolytic activation of diverse ligands of the EGF receptor.
One human gene sharing homology with multiple cDNA sequences (Accession No. AK026010, NM—022450, Z69719, AK056708, BC014425, M99624) has been annotated as an ortholog of mouse epidermal growth factor receptor related sequence (EGFR-RS), hypothetical protein similar to epidermal growth factor receptor-related protein, human epidermal growth factor receptor-related gene, and lately human rhomboid family 1. The cDNA sequences AK026010, BC014425 and NM—022450 encode the same 855 amino acid protein (Accession No. BAB15318, AAH14425 and AAA02490). However, the biological activity of this protein presently is unknown.
TRA1 (Validated Target ICT1025): Homo sapiens Tumor rejection antigen, TRA1 or heat shock protein gp96 or grp94 is published with GenBank accession nos. NM—003299, AK025459, BC009195, AY040226, X15187 and AF087988. See also, U.S. Publication Nos. 2003/0215874; 2003/0054996; and 2002/0160496.
One of the targets selected with Efficacy-First, tumor rejection antigen-1 (TRA-1), was found to have increased expression in tumors induced to accelerated growth. TRA-1, also known as glucose-regulated protein 94 (grp94), gp96, endoplasmiin precursor and other names, was first described as a molecular chaperone [Hartl FU. (1996) Molecular chaperones in cellular protein folding. Nature 381(6583):571-9] with important roles in endoplasmic reticulum related to nuclear signaling, protein folding, sorting and secretion [Nicchitta, C. V. (1998): Biochemical, cell biological and immunological issues surrounding the endoplasmic reticulum chaperone GRP94/gp96. Current Opinion in Immunology, 10:103-109.]. In addition, it exerts a specific protection against Ca2+ depletion stress and is involved in antigen presentation [Tamura, Y. P. Peng, K. Liu, M. Daou, P. K. Srivastava, 1997: Immunotherapy of tumors with autologous tumor-derived heat shock protein preparation. Science, 278:117-120]. Furthermore, it also has an important role in tumorigencity [Udono H, Levey D L, Srivastava P K. (1994) Cellular requirements for tumor-specific immunity elicited by heat shock proteins: tumor rejection antigen gp96 primes CD8=T cells in vivo. Pro Natl Acad Sci USA 91: 3077-3081.]. Menoret et al. [Menoret A, Meflah K, Le Pendu J. (1994) Expression of the 100 kDa glucose-regulated protein (GRP100/endoplasmin) is associated with tumorigenicity in a model of rat colon adenocarcinoma. Int J Cancer 56: 400-405] reported that there was an overexpression of TRA-1 in a model of rat colon adenocarcinoma. Gazit et al. [Gadi Gazit, Jun lu, Amy S. Lee. (1999) De-regulation of GRP stress protein expression in human breast cancer cell lines. Breast Cancer Research and Treatment 54: 135-146.] found out there was a 3-5 fold increase in the level of TRA-1 protein was observed in five human breast cancer lines as compared to the normal human mammary lines. Cai et al. [Cai J W. Henderson B W, Shen J W, et al (1993) Induction of glucose-regulated proteins during growth of murine tumor. J Cell Physiol 154; 229-237] found through studies during growth of tumors that the level of the TRA-1 is increased, correlating with the size of the tumor. Elevated level of TRA-1 has been implicated to protect neoplastic cells and tumors against cytotoxic T-lymphocyte mediated cytotoxicity and protected tissues culture cells against adverse physiological conditions [Sugawara S, Takeda K, Lee A, et al. (1993) Suppression of stress protein GRP78 induction in tumor B/C10ME eliminates resistance to cell mediated cytotoxicity. Cancer Research. 53: 6001-6005]. Public domain databases reveal that TRA-1 is over-expressed in many human cancer tissues including prostate, mammary, brain, stomach, and soft tissue tumors. Overexpression, antisense and ribozyme approaches in tissue culture system directly showed that TRA-1 could protect cells against cell death [Little E, Ramakrishnan M, Roy B, et al. (1994) The glucose-regulated proteins (GRP78 and GRP94): Functions, gene regulation, and applications. Crit Rev Eukaryot Gene Expr 4: 1-18, Garrido C, Gurbuxani S, Ravagnan L, Kroemer G. (2001). Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun. 286(3):433-42., Ramachandra K. Reddy, et al. (1999). The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca2+-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis. J Biol. Chem 274: 28476-28483]. These anti-apoptosis effects of TRA-1 are associated with induction in neoplastic cells and may lead to cancer progression and chemotherapy resistance. Although normally confined to the ER, TRA-1 has been shown to escape to KDEL (SEQ ID NO: 103)-mediated retention system in several cell types. For instance, a significant fraction of TRA-1 is secreted to the extracellular space by hepatocytes and exocrine pancreatic cells, via the normal secretory pathway. In several tumor cell lines TRA- 1 is detectable as an outer surface protein [Altmeyer A, Maki R G, Feldweg A M, Heike M, Protopopov V P, Masur S K, Srivastava P K (1996). Tumor-specific cell surface expression of the-KDEL (SEQ ID NO: 103) containing, endoplasmic reticular heat shock protein gp96. Int. J Cancer 22;69(4):340-9.].
TRA-1 has been shown to chaperone a broad array of peptides, including those derived from normal proteins as well as from foreign and altered proteins present in cancer or virus-infected cells. Thus, tumor-derived TRA-1 carries tumor antigenic peptides, and its preparations from virus-infected cells carry viral epitopes. Although TRA-1 is normally intracellular, necrotic cells release TRA-1 peptide complexes, which are taken up by scavenging antigen-presenting cells. Presentation of the peptides on the surface of these cells leads to stimulation of T lymphocytes and a pro-inflammatory response.
Complexes of TRA-1 with peptides, whether isolated from cells or reconstituted in vitro, have been demonstrated to serve as effective vaccines, producing anti-tumor immune responses in animals and in man [Tamura, Y. P. Peng, K. Liu, M. Daou, P. K. Srivastava, 1997: Immunotherapy of tumors with autologous tumor-derived heat shock protein preparation. Science, 278:117-120.]. Oncophage is a vaccine made from individual patients' tumors. Patients have surgery to remove part or all of the cancerous tissue, and a portion of this tissue is shipped overnight to Antigenics' manufacturing facility in Massachusetts. The Oncophage clinical studies in several cancers including pancreatic, melanoma, kidney, colorectal, gastric, and non-Hodgkin's lymphoma have yielded very promising results. Their analysis provides a strong indication that antigen presentation by TRA-1 can induce an immune response in patients and clinical responses. With melanoma or colorectal cancer in one study, 10 out of 39 melanoma patients responded clinically to Oncophage treatment, including two patients whose cancer disappeared completely for more than two years. Of the 24 melanoma patients who were evaluated for immune response, 10 demonstrated increased antimelanoma T-cell activity. In colorectal cancer patients, a T-cell response was observed in 17 out of 29 patients, and seemed to be correlated with survival. The mechanism by which Oncophage induces immune response in melanoma and colorectal cancer was determined to be the sam-—confirming a wealth of preclinical and early clinical data demonstrating that this mechanism is virtually identical in all cancers and species tested to date.
MFGE8 (Validated Target ICT1030): Homo sapiens milk fat globule-EGF factor 8 protein (MFGE8) or breast epithelial BA46 antigen is published under GenBank accession nos. is NM—005928 and BC003610. U.S. Pat. No. 6,339,066 B1 describes aspects of MFGE8 related molecules such as ‘protein kinase C-eta’ (PKC-η).
TNFSF13 (Validated Target ICT1031): Homo sapiens Tumor necrosis factor ligand super family member 13 (TNFSF13) is published GenBank accession nos. are AK090698 and O75888. Several international patent applications describe aspects of TNFSF13 related molecules such as APRIL (A proliferation-inducing ligand), TALL-2 (TNF-and APOL-related leukocyte expressed ligand 2), and TRDL- 1 (TNF-related death ligand-1) (see, for example, WO 99/12965 and WO 01/60397).
ZFP236 (Validated Target ICT1003): Homo sapiens zinc finger protein 236 (ZFP236) is published under GenBank accession no. AK000847.